Research pertaining to this grant has evolved toward determining the role of microglia in mediating ethanol apoptotic action on developing R-endorphin neurons involved in regulation ofthe hypothalamic pituitary adrenal axis (HPA) functions. The work conducted by us during the past funding period of the MERIT award demonstrated that alcohol exposure during early life causes activation of microglial cells and releases cytokines and chemokines that impact the physiological functions of hypothalamic neurons. Furthermore, early life exposure of ethanol may program the microglial cell population to produce more inflammatory cytokines following a stress challenge in the adulthood. Also, B-endorphin neurons that regulate the HPA axis are not only a target of inflammatory cytokines and chemokines but also a modulator of microglial cell functions. Given the lack of information how microglia and BEP interact to control stress responses and the availability of methods of culturing pure B-endorphin neurons from rat neuronal stem cells, we propose to test the following hypotheses: i) alcohol exposures stimulate microglia to produce inflammatory cytokines including TNF-a that activates the nuclear factor-KB (NFKB) pathway and NADPH oxidase to induce apoptotic signaling in developing B-endorphin neurons; ii) li-endorphin neurons influences the ethanol's ability to increase inflammatory cytokines production by altering opioid receptor functions in microglial cells; and iii) early-life alcohol exposures program the microglial cell population to produce more inflammatory cytokines following a stress challenge during the adulthood. We will employ various state-of-the art techniques involving in vitro differentiated pure B-endorphin cells, primary cultures of microglial cells, neonatal model of alcohol feeding, microarray and proteomic approaches, western blot, realtime PCR, MeDIP assay, double immunocytochemistry, gene knocking-down. We will also use both in vitro and in vivo approaches. We anticipate these studies will identify whther a bi-directional communication between microglia and B-endorphin within the hypothalamus may predict risk for stress abnormality in a model of developmental alcohol exposures. RELEVANCE (See instructions): The proposed series of studies should continue to generate valuable data for better understanding pf ethanol's neurotoxic action on developing B-endorphin neurons. Additionally, the proposed research should identify compounds acting on novel targets to inhibit the release of a wide range of proinflammatory factprs from overactivated microglia that might be critical for preventing of B-endorphin neuronal death.